Neuropeptide Neuromedin Research Articles

A Nociceptive Signaling Role for Neuromedin B

Here we used an array-based differential screen to uncover the expression of the neuropeptide neuromedin B (NMB) in the trigeminal ganglia of mice. Double-labeling experiments reveal NMB is expressed in a subset of sensory neurons that colabel with calcitonin gene-related peptide and TRPV1 suggestive of a role for NMB in nociception. Indeed, administration of NMB antagonist greatly attenuates edema and nerve sensitization following stimulation of peripheral nerves with mustard oil, demonstrating that NMB contributes to neurogenic inflammation. Moreover, direct injection of NMB causes local swelling and nociceptive sensitization. Interestingly, we also find that the receptor for NMB is expressed in interneurons in the superficial layers of the dorsal horn. We used NMB-saporin to specifically eliminate NMBR-expressing neurons and determined they are required in responses to noxious heat, but not for reaction to mechanical and pruritic stimuli. Thus, NMB may be a neurotransmitter that is selectively involved in the perception of thermal stimuli.

The c-Myb target gene neuromedin U functions as a novel cofactor during the early stages of erythropoiesis

AbstractThe requirement of c-Myb during erythropoiesis spurred an interest in identifying c-Myb target genes that are important for erythroid development. Here, we determined that the neuropeptide neuromedin U (NmU) is a c-Myb target gene. Silencing NmU, c-myb, or NmU's cognate receptor NMUR1 expression in human CD34+ cells impaired burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) formation compared with control. Exogenous addition of NmU peptide to NmU or c-myb siRNA-treated CD34+ cells rescued BFU-E and yielded a greater number of CFU-E than observed with control. No rescue of BFU-E and CFU-E growth was observed when NmU peptide was exogenously added to NMUR1 siRNA-treated cells compared with NMUR1 siRNA-treated cells cultured without NmU peptide. In K562 and CD34+ cells, NmU activated protein kinase C-βII, a factor associated with hematopoietic differentiation-proliferation. CD34+ cells cultured under erythroid-inducing conditions, with NmU peptide and erythropoietin added at day 6, revealed an increase in endogenous NmU and c-myb gene expression at day 8 and a 16% expansion of early erythroblasts at day 10 compared to cultures without NmU peptide. Combined, these data strongly support that the c-Myb target gene NmU functions as a novel cofactor for erythropoiesis and expands early erythroblasts.

Mechanisms of Appetite Regulation

Mechanisms of Appetite Regulation

Increased Transcription of the Neuropeptide Neuromedin U by Ets-2 and C-Myb Not B-Myb in Human Myeloid Leukemia Cells Promotes Their Proliferation.

Neuromedin U (NmU) is a 25-amino acid neuropeptide that is expressed at high levels in the brain, gut, and bone marrow. In the brain, NmU modulates the central control of feeding and bone mass. In the intestine, NmU regulates the contraction of smooth muscle, local blood flow, and ion transport. In the bone marrow, the physiological function of NmU remains ill-defined. To determine the physiological function of NmU in hematopoietic cells, we utilized K562-MERT cells, which express a tamoxifen-inducible dominant negative Myb (MERT), and cell culture assays. We chose to test the function of NmU in a cell line that expresses high levels of the proto-oncogene c-myb because c-Myb encodes a transcription factor that regulates cell proliferation, survival, and differentiation through the transactivation of its target genes. By inhibiting endogenous Myb activity in K562-MERT cells by tamoxifen in methylcellulose cultures, we observed a 6-fold decrease in cell number compared to untreated K562-MERT cells. Supplementing the tamoxifen treated methylcellulose cultures of K562-MERT cells with NmU resulted in a 3.6-fold increase in K562-MERT cell number compared to K562-MERT cells treated with only tamoxifen. In liquid cultures using primary cells from patients with acute myeloid leukemia, we observed more cells in the cultures with NmU than when NmU was absent from the cultures. Finally, silencing NmU gene expression in K562 cells via RNA interference decreased the proliferation of these cells. Collectively, these data demonstrate that NmU functions to promote the proliferation of hematopoietic cells. The ability of NmU to rescue cell growth in tamoxifen treated K562-MERT cells and Myb's role in regulating hematopoietic cell proliferation led us to hypothesize that Myb mediates hematopoietic cell proliferation in part by directly regulating NmU gene expression. To test this hypothesis, we examined the DNA sequence upstream of NmU's predicted transcription start site (as noted in Genbank accession #NM_006681) for potential Myb response elements (MREs). We identified eleven potential MREs within the first 2kb upstream of NmU's transcription start site. Of these MREs, five were identified as canonical (PyAAC(G/T)G). Our search also identified potential Ets-2 binding motifs within the human NmU promoter region, which were of interest because Myb has been reported to cooperate with Ets-2 in the regulation of c-kit and CD34 gene expression. To determine if any of the potential MREs within the NmU promoter were functional, we first completed in vitro assays using luciferase reporter constructs followed by in vivo assays using chromatin immunoprecipitation (ChIP) assays. The luciferase reporter constructs were generated such that the first 2kb upstream of NmU's transcription start site was inserted upstream of the luciferase gene in pGL3-basic to yield pGL3-NmU. When pGL3-NmU was co-transfected into 293T cells with a c-Myb expression construct, we observed an average of 15-fold induction of luciferase activity compared to empty vector. There was no change in luciferase activity when 293T cells were co-transfected with the c-Myb isoform B-Myb compared to empty vector, suggesting that c-Myb and not B-Myb interact with the MREs within the NmU promoter to induce its expression. Mutation of either MRE9 or 10, which are distal to NmU's transcription start site, in pGL3-NmU prevented c-Myb from inducing luciferase gene expression, demonstrating that MRE9 and 10 are functional. When Ets-2 was co-transfected with pGL3-NmU into 293T cells, a 10-fold increase in luciferase activity was observed suggesting that Ets-2 and c-Myb may cooperate to increase the transcription of NmU. To determine the physiologic relevance of our in vitro luciferase studies, we performed ChIP assays. Following immunoprecipitation of the cross-linked chromatin with either anti-c-Myb or anti-Ets-2, a PCR product of the recovered DNA was observed using primers that flanked MRE9, MRE10, and one Ets-2 site, demonstrating that in vivo c-Myb and Ets-2 interact directly with NmU's promoter. Studies are underway to determine whether c-Myb and Ets-2 cooperate to induce NmU gene expression. Further characterization of the regulation of NmU gene expression in normal and malignant hematopoietic cells may yield new clues to Myb's role in leukemogenesis and could suggest new therapeutic targets in human myeloid leukemia cells.

Neuromedin U stimulates contraction of human long saphenous vein and gastrointestinal smooth muscle in vitro

The neuropeptide Neuromedin U (NMU) stimulates smooth muscle contraction, and modulates local blood flow and adrenocortical function via two endogenous receptors, NMU1 and NMU2. Although its amino-acid sequence is highly conserved across species, the physiological effects of NMU are variable between species and little is known of its effects on human tissues. We have examined the contractile effects of NMU-25 on human smooth muscles of the gastrointestinal (GI) tract (ascending colon, gallbladder) and long saphenous vein (LSV) using in vitro organ bath bioassays. From LSV, ileum, gallbladder, caecum and colon, NMU receptor transcripts were amplified by RT-PCR and expression levels were determined by semi-quantitative scanning densitometry. NMU-25 produced a concentration-dependent, sustained contraction of isolated smooth muscle ( p[ A] 50 ± s.e.m., ascending colon, 8.93 ± 0.18; gallbladder, 7.01 ± 0.15; LSV, 8.67 ± 0.09). NMU1 and NMU2 receptor transcription was detected in all tissues; transcription of both receptors was similar in gallbladder, but NMU1 receptor transcription was predominant in the sigmoid colon and LSV. In summary, these studies indicate that NMU may control tone in the human GI tract and LSV through an action on smooth muscle. Development of NMU receptor subtype-selective ligands will aid the further elucidation of the physiological roles of NMU and its two receptors.

The neuropeptide neuromedin U activates eosinophils and is involved in allergen-induced eosinophilia

Neuromedin U (NMU) is a neuropeptide expressed not only in the central nervous system but also in various organs, including the gastrointestinal tract and lungs. NMU interacts with two G protein-coupled receptors, NMU-R1 and NMU-R2. Although NMU-R2 is expressed in a specific region of the brain, NMU-R1 is expressed in various peripheral tissues, including immune and hematopoietic cells. Our recent study demonstrated an important role of NMU in mast cell-mediated inflammation. In this study, we showed that airway eosinophilia was reduced in NMU-deficient mice in an allergen-induced asthma model. There were no differences in the antigen-induced Th2 responses between wild-type and NMU knockout mice. NMU-R1 was highly expressed in the eosinophil cell line, and NMU directly induced Ca(2+) mobilization and extracellular/signal-regulated kinase phosphorylation. NMU also induced cell adhesion to components of the extracellular matrix (fibronectin and collagen type I), and chemotaxis in vitro. Furthermore, NMU-R1 was also expressed in human peripheral blood eosinophils, and NMU induced cell adhesion in a dose-dependent manner. These data indicate that NMU promotes eosinophil infiltration into inflammatory sites by directly activating eosinophils. Our study suggests that NMU receptor antagonists could be novel targets for pharmacological inhibition of allergic inflammatory diseases, including asthma.

Neuromedin U Partially Mediates Leptin-Induced Hypothalamo-Pituitary Adrenal (HPA) Stimulation and Has a Physiological Role in the Regulation of the HPA Axis in the Rat

Intracerebroventricular (ICV) administration of the hypothalamic neuropeptide neuromedin U (NMU) or the adipostat hormone leptin increases plasma ACTH and corticosterone. The relationship between leptin and NMU in the regulation of the hypothalamo-pituitary adrenal (HPA) axis is currently unknown. In this study, leptin (1 nm) significantly increased the release of CRH from ex vivo hypothalamic explants by 207 +/- 8.4% (P < 0.05 vs. basal), an effect blocked by the administration of anti-NMU IgG. The ICV administration of leptin (10 mug, 0.625 nmol) increased plasma ACTH and corticosterone 20 min after injection [plasma ACTH (picograms per milliliter): vehicle, 63 +/- 20, leptin, 135 +/- 36, P < 0.05; plasma corticosterone (nanograms per milliliter): vehicle, 285 +/- 39, leptin, 452 +/- 44, P < 0.01]. These effects were partially attenuated by the prior administration of anti-NMU IgG. Peripheral leptin also stimulated ACTH release, an effect attenuated by prior ICV administration of anti-NMU IgG. We examined the diurnal pattern of hypothalamic NMU mRNA expression and peptide content, plasma leptin, and plasma corticosterone. The diurnal changes in hypothalamic NMU mRNA expression were positively correlated with hypothalamic NMU peptide content, plasma corticosterone, and plasma leptin. The ICV administration of anti-NMU IgG significantly attenuated the dark phase rise in corticosterone [corticosterone (nanograms per milliliter): vehicle, 493 +/- 38; NMU IgG, 342 +/- 47 (P < 0.05)]. These studies suggest that NMU may play a role in the regulation of the HPA axis and partially mediate leptin-induced HPA stimulation.

The neuropeptide neuromedin U promotes IL-6 production from macrophages and endotoxin shock

Neuromedin U (NMU) is a neuropeptide involved in appetite, circadian rhythm, and pronociception. However, the NMU receptor NMU-R1 has been shown to be expressed in immune cells and NMU promotes mast cell-dependent inflammation. In this study, we demonstrated that NMU plays an important role in IL-6 production in macrophages. NMU-deficient mice were resistant against cecal ligation puncture- as well as LPS-induced septic shock. IL-6 but not TNF-α levels were markedly reduced in LPS-treated NMU-deficient mice compared with wild type mice. Both NMU and NMU-R1 were expressed in wild type peritoneal macrophages, and treatment with LPS resulted in up-regulation of NMU but down-regulation of NMU-R1 expression, however, no down-regulation of NMU-R1 was observed in NMU-deficient macrophages where LPS-induced IL-6 production was severely reduced. These data suggest that LPS-induced IL-6 expression is partly dependent on autocrine/paracrine activation of the NMU–NMU-R1 signals in macrophages.

The neuropeptide neuromedin U promotes inflammation by direct activation of mast cells

Neuromedin U (NMU) is a neuropeptide that is expressed in the gastrointestinal tract and central nervous system. NMU interacts with two G protein–coupled receptors, NMU-R1 and NMU-R2. Whereas NMU-R2 localizes predominantly to nerve cells, NMU-R1 is expressed in peripheral tissues including lymphocytes and monocytes, suggesting a role of NMU in immunoregulation. However, the functions of NMU in peripheral tissues have not been clarified. In this study, using NMU-deficient mice, we first demonstrated that NMU plays an important role in mast cell-mediated inflammation. Complete Freund's adjuvant-induced mast cell degranulation as well as edema and neutrophil infiltration, which occurred weakly in mast cell–deficient WBB6F1-W/W v mice, did not occur in NMU-deficient mice. Moreover, intraplantar injection of NMU into paws induced early inflammatory responses such as mast cell degranulation, vasodilation, and plasma extravasation in WT mice but not in WBB6F1-W/W v mice. NMU-R1 was highly expressed in primary mast cells, and NMU induced Ca2+ mobilization and degranulation in peritoneal mast cells. These data indicate that NMU promotes mast cell–mediated inflammation; therefore, NMU receptor antagonists could be a novel target for pharmacological inhibition of mast cell–mediated inflammatory diseases.

Signaling and ligand binding by recombinant neuromedin U receptors: evidence for dual coupling to Galphaq/11 and Galphai and an irreversible ligand-receptor interaction.

The neuropeptide neuromedin U (NmU) shows considerable structural conservation across species. Within the body, it is widely distributed and in mammals has been implicated in physiological roles, including the regulation of feeding, anxiety, pain, blood flow, and smooth muscle contraction. Human NmU-25 (hNmU-25) and other NmU analogs were recently identified as ligands for two human orphan G protein-coupled receptors, subsequently named hNmU-R1 and hNmU-R2. These receptors have approximately 50% amino acid homology, and, at least in mammalian species, NmU-R1 and NmU-R2 are expressed predominantly in the periphery and central nervous system, respectively. Here, we have characterized signaling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in human embryonic kidney 293 cells, particularly to define their G protein coupling and the activation and regulation of signal transduction pathways. We show that these receptors couple to both Galpha(q/11) and Galpha(i). Activation of either receptor type causes a pertussis toxin-insensitive activation of both phospholipase C and mitogen activated-protein kinase and a pertussis toxin-sensitive inhibition of adenylyl cyclase with subnanomolar potency for each. Activation of phospholipase C is sustained, but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not cause repetitive intracellular Ca2+ signaling by either recombinant receptors or those expressed endogenously in isolated smooth muscle cells from rat fundus. Using several strategies, we show this to be a consequence of essentially irreversible binding of hNmU-25 to its receptors and that this is followed by ligand internalization. Despite structural differences between receptors, there were no apparent differences in their activation, coupling, or regulation.

Pro-nociceptive effects of neuromedin u in rat

The neuropeptide neuromedin U (NMU) has been shown to have significant effects on cardiovascular, gastrointestinal and CNS functions. The peptide was first isolated from the porcine spinal cord and later shown to be present in spinal cords of other species. Little is known about the distribution of neuromedin U receptors (NMURs) in the spinal cord and the spinal action of the peptide. Here we report on the expression of NMURs and a potential role in nociception in the rat spinal cord using a combination of behavioral and electrophysiological studies. Receptor autoradiography showed that NMU-23 binding was restricted to the superficial layers of spinal cord, a region known to be involved in the control of nociception. In situ hybridization analysis indicated the mRNA of NMUR2 was located in the same region (laminae I and IIo) as NMU-23 binding, while the mRNA for NMU receptor 1 was observed in a subpopulation of small diameter neurons of dorsal root ganglia. Intrathecal (i.t.) administration of neuromedin U-23 (0.4–4.0 nmol/10 μl) dose-dependently decreased both the mechanical threshold to von Frey hair stimulation and the withdrawal latency to a noxious thermal stimulus. Mechanical allodynia was observed between 10 and 120 min, peaking at 30 min and heat hyperalgesia was observed 10–30 min after i.t. administration of NMU-23. A similar mechanical allodynia was also observed following i.t. administration of NMU-8 (0.4–4 nmol/10 μl). A significant enhancement of the excitability of flexor reflex was induced by intrathecal administration of NMU-23 (4 nmol/10 μl). Evoked responses to touch and pinch stimuli were increased by 439±94% and 188±36% ( P<0.01, n=6) respectively. The behavioral and electrophysiological data demonstrate, for the first time, a pro-nociceptive action of NMU. The restricted distribution of NMU receptors to a region of the spinal cord involved in nociception suggests that this peptide receptor system may play a role in nociception.

A pro-nociceptive role of neuromedin U in adult mice

Although the neuropeptide neuromedin U (NMU) was first isolated from the spinal cord, its actions in this site are unknown. The recent identification of the NMU receptor subtype 2 (NMU2R) in the spinal cord has increased the interest in investigating the role of NMU in this part of the central nervous system. Here, we report a novel function for NMU in spinal nociception in the mouse. Systemic perfusion of NMU (rat, NMU-23) dose-dependently (0.2, 0.5, 1, and 2.5 μM) potentiated both the background activity and noxious pinch-evoked response of nociceptive or wide dynamic range, but not non-nociceptive, dorsal horn neurons. At 2.5 μM, NMU-23 increased the total background activity from 154±34 to 1374±260 spikes/160 s ( P<0.005, n=28) and increased the evoked nociceptive response by 185±50% ( P<0.01, n=13). Intrathecal administration of NMU-23 (0.4, 1.1, and 3.8 nmol/10 μl) dose-dependently decreased thermal withdrawal latencies and produced a morphine-sensitive behavioral response. These electrophysiological and behavioral results suggest that NMU may be a novel physiological regulator in spinal nociceptive transmission and processing.

Conformational Changes of Neuromedin B and Delta Sleep-Inducing Peptide Induced by Their Interaction with Lipid Membranes as Revealed by Spectroscopic Techniques and Molecular Dynamics Simulation

Static and dynamic spectroscopic properties of the tryptophanil emission in conjunction with circular dichroism (CD) spectroscopy and molecular dynamics are used to investigate the interactions of the neuropeptide neuromedin B (NMB) and the membrane-permeable δ sleep-inducing peptide (DSIP) with the membrane lipid phase. Our data indicate that in solution both peptides exist in energetically equivalent conformations, whereas in the presence of the membrane specific conformational states are stabilized. By changing from the aqueous to the lipid phase, the static and the dynamic fluorescence properties of the NMB's tryptophan residue are clearly affected: the fluorescence steady-state spectrum as well as the resolved fluorescence decay-associated spectra (DAS) are shifted to the blue with a significant increase of the fluorescence intensity of the second lifetime component (τ2-DAS). On the other hand, in the lipid environment the same parameters of DSIP are negligibly affected as compared to the aqueous buffer. The CD and molecular dynamics analyses are consistent with these results and indicate that, while NMB assumes a helix-like conformation with the tryptophan residue in the apolar surface, DSIP adopts a globule-like structure with the indole ring that is surface-exposed. As previously found for neuromedin C (Polverini, E., Neyroz, P., Fariselli, P., Casadio, R., and Masotti, L.,Biochem. Biophys. Res. Commun.214, 663–668, 1995), for NMB the stabilized “lipophilic” structure also may favor the correct peptide–receptor contact and recognition. For DSIP, the lipid-stabilized conformation does not support an amphiphilic structure-driven peptide–membrane interaction and suggests a hydrophobicity-driven diffusion across the bilayer.